Our long-term goal is to understand how ion channels in the retinal pigment epithelium (RPE) operate to maintain the appropriate extracellular environment required for photoreceptor health and integrity. Ionic homeostasis of the subretinal space (SRS) is achieved by the transport of various ions across the RPE. This transport entails the coordinated activity of a diverse group of ion transporter and ion channel proteins residing in the RPE apical and basolateral membranes. The importance of RPE ion transport is underscored by the fact that mutations in genes encoding ion channels expressed in the RPE cause inherited retinal degenerations. We have discovered that RPE cells have a previously unrecognized ion channel that is remarkably selective for the biologically active anion, thiocyanate (SCN-). SCN- is of interest because it is present in most extracellular fluids and is known to play key roles in innate immunity, redox regulation, and synaptic modulation. Based on our new findings, we posit that a key gap in our understanding of the RPE is its role in the transport of the SCN- out of the SRS. The overall objective of this competing-renewal project is to characterize the cellular mechanisms that enable the RPE to perform transepithelial SCN- transport. Our central hypothesis is that the RPE is endowed with an asymmetric distribution of specialized ion channels and transporters that operate together to actively transport SCN- between the SRS and choriocapillaris. The rationale that underlies this proposal is that delineation of the SCN- transport mechanisms in the RPE is crucial for understanding how the SCN- concentration in the SRS is regulated. This is important because elevation of the plasma SCN- level, a consequence of smoking, may alter the SCN- concentration in the SRS to potentially damage photoreceptors. The specific aims of this proposal are to: (1) test hypotheses about specific mechanisms that transport SCN- across the RPE apical and basolateral membranes; (2) test the expression of candidate SCN- channel and transporter genes in the RPE and ascertain the subcellular distribution of the encoded proteins; and (3) determine the direction and magnitude of active SCN- transport across the RPE and ascertain the involvement of candidate SCN- channels and transporters. With respect to expected outcomes, the proposed work is expected to functionally characterize and identify the components of a novel SCN- transport system in the RPE. The impact of this contribution is significant because it will provide insights into the molecular mechanisms that regulate the photoreceptor environmental level of SCN-, a biologically active molecule that is known to have both positive and negative effects elsewhere in the body. The proposed research is innovative because it will determine for the first time how a previously unknown ion channel and other membrane proteins are functionally organized to coordinate the active transport of SCN- across the RPE.